We have isolated purely rat sarcoma viruses (RaSV) after separate cocultivation of rat leukemia virus (RaLV) productive Sprague Dawley rat cells (SD-1T) with chemically or polyoma virus transformed rat cultures. The RaSV was not isolated when these same cells were cocultivated with cultures producing other RaLV strains or murine, feline or baboon type C helper viruses. Thus, the probability of rat sarcoma virus rescue in vitro by any mammalian type C virus is extremely low. We also tested 21 additional rat tumor cell lines that were transformed by chemical carcinogens or by various DNA viruses including SV40 and Herpes. None rescued RaSV when cocultivated with SD-1T cells. Analysis of the biological properties of the 25 different transformed tumor cells failed to identify unique features of these four cells that were conducive for RaSV rescue. Only one criteria that appears to be commonly shared between the four rescuable cells is the numerous serial passages in rats. This indicates that during in vivo serial transplantation the src genes are enriched in a manner that they can be rescued by an endogenous resplicating virus. The RaSV transformed virus producer and nonproducer cells both show a phosphoprotein of 29,000 daltons (P29). This is distinct but immunologically related to a 21,000 dalton phosphoprotein of the mouse-rat recombinant viruses, i.e., Kirsten and Harvey strains of murine sarcoma viruses. In collaboration with Dr. H. Young (Frederick Cancer Research Center) we are chacterizing different rat and heterologous species' cells for the expression of this protein. We are also attempting to isolate mutants of various rat cells and RaSV isolates. This would help in understanding the mechanism of rat sarcoma virus rescue in vitro.